BRCA2 associates with MCM10 to suppress PRIMPOL-mediated repriming and single-stranded gap formation after DNA damage.

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

The anti-parasitic agent suramin and several of its analogues are inhibitors of the DNA binding protein Mcm10.

Minichromosome maintenance protein 10 (Mcm10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit Mcm10 from binding to DNA. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human Mcm10 internal domain and full-length Xenopus Mcm10, including analogues that are selective for Mcm10 over human RPA. Binding of suramin analogues to Mcm10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human Mcm10 binding affinity (FP Ki 170 nM, SPR KD 460 nM) and cell activity (IC50 38 µM). Suramin and its analogues are the first identified inhibitors of Mcm10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.

Inhibiting the MCM8-9 complex selectively sensitizes cancer cells to cisplatin and olaparib.

MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.

TGFß1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-Deficient Conditions.

Transforming growth factor ß1 (TGFß1) is a potent inhibitor of cell growth that targets gene-regulatory events, but also inhibits the function of CDC45-MCM-GINS helicases (CMG; MCM, Mini-Chromosome Maintenance; GINS, Go-Ichi-Ni-San) through multiple mechanisms to achieve cell-cycle arrest. Early in G1, TGFß1 blocks MCM subunit expression and suppresses Myc and Cyclin E/Cdk2 activity required for CMG assembly, should MCMs be expressed. Once CMGs are assembled in late-G1, TGFß1 blocks CMG activation using a direct mechanism involving the retinoblastoma (Rb) tumor suppressor. Here, in cells lacking Rb, TGFß1 does not suppress Myc, Cyclin E/Cdk2 activity, or MCM expression, yet growth arrest remains intact and Smad2/3/4-dependent. Such arrest occurs due to inhibition of MCM hexamer assembly by TGFß1, which is not seen when Rb is present and MCM subunit expression is normally blocked by TGFß1. Loss of Smad expression prevents TGFß1 suppression of MCM assembly. Mechanistically, TGFß1 blocks a Cyclin E-Mcm7 molecular interaction required for MCM hexamer assembly upstream of CDC10-dependent transcript-1 (CDT1) function. Accordingly, overexpression of CDT1 with an intact MCM-binding domain abrogates TGFß1 arrest and rescues MCM assembly. The ability of CDT1 to restore MCM assembly and allow S-phase entry indicates that, in the absence of Rb and other canonical mediators, TGFß1 relies on inhibition of Cyclin E-MCM7 and MCM assembly to achieve cell cycle arrest. IMPLICATIONS: These results demonstrate that the MCM assembly process is a pivotal target of TGFß1 in eliciting cell cycle arrest, and provide evidence for a novel oncogenic role for CDT1 in abrogating TGFß1 inhibition of MCM assembly.

Destabilization of the MiniChromosome Maintenance (MCM) complex modulates the cellular response to DNA double strand breaks.

DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.

Interaction between RAD51 and MCM Complex Is Essential for RAD51 Foci Forming in Colon Cancer HCT116 Cells.

Colon cancer remains one of the most common digestive system malignancies in the World. This study investigated the possible interaction between RAD51 and minichromosome maintenance proteins (MCMs) in HCT116 cells, which can serve as a model system for forming colon cancer foci. The interaction between RAD51 and MCMs was detected by mass spectrometry. Silenced MCM vectors were transfected into HTC116 cells. The expressions of RAD51 and MCMs were detected using Western blotting. Foci forming and chromatin fraction of RAD51 in HCT116 cells were also analyzed. The results showed that RAD51 directly interacted with MCM2, MCM3, MCM5, and MCM6 in colon cancer HTC116 cells. Suppression of MCM2 or MCM6 by shRNA decreased the chromatin localization of RAD51 in HTC116 cells. Moreover, silenced MCM2 or MCM6 decreased the foci forming of RAD51 in HTC116 cells. Our study suggests that the interaction between MCMs and RAD51 is essential for the chromatin localization and foci forming of RAD51 in HCT116 cell DNA damage recovery, and it may be a theoretical basis for analysis of RAD51 in tumor samples of colon cancer patients.

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.

Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation.

Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase ε 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.

HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest.

Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.

Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction.

Mitochondrial DNA (mtDNA) encodes proteins that are important for ATP biogenesis. Therefore, changes in mtDNA copy number will have profound consequences on cell survival and proliferation. RECQ4 DNA helicase participates in both nuclear DNA and mtDNA synthesis. However, the mechanism that balances the distribution of RECQ4 in the nucleus and mitochondria is unknown. Here, we show that RECQ4 forms protein complexes with Protein Phosphatase 2A (PP2A), nucleophosmin (NPM), and mitochondrial p32 in different cellular compartments. Critically, the interaction with p32 negatively controls the transport of both RECQ4 and its chromatin-associated replication factor, MCM10, from the nucleus to mitochondria. Amino acids that are deleted in the most common cancer-associated RECQ4 mutation are required for the interaction with p32. Hence, this RECQ4 mutant, which is no longer regulated by p32 and is enriched in the mitochondria, interacts with the mitochondrial replication helicase PEO1 and induces abnormally high levels of mtDNA synthesis.

Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase.

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2-7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.